Morinaga Milk Industry Co, Ltd, Zama, Kanagawa, Japan.
J Food Prot. 2012 Sep;75(9):1572-9. doi: 10.4315/0362-028X.JFP-12-015.
The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMA-DqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.
本研究的目的是建立一种快速检测方法，用于特异性检测婴儿配方粉（PIF）中的活克罗诺杆菌（Cronobacter sakazakii）。在 PCR 之前，用浓度梯度（gEMA）的吖啶橙对样品进行多次处理，以区分活的和死的 C. sakazakii 细胞。为了提高当前的检测限，我们开发了一种新的缓冲液，用于无需 DNA 分离的直接定量实时 PCR（DqPCR）。使用 17 个 PIF 样品，我们的快速检测方法与 2012 年《细菌分析手册》中公布的新美国食品和药物管理局（FDA）方法进行了比较。尽管新的 FDA 方法和我们的快速检测方法（DqPCR 与 gEMA 联合使用，gEMA-DqPCR）都对 17 个 PIF 样本均产生阴性结果，但未经 EMA 处理的 17 个 PIF 中有 5 个为 DqPCR 阳性。此外，对于人工污染有活的 C. sakazakii 的 PIF 样品，gEMA-DqPCR 可在 9 小时内成功检测到 300 g PIF 中 1 至 9 CFU 的活 C. sakazakii，包括 6 小时的预孵育。我们的结果表明，由于商业 PIF 中存在死的或受伤的 C. sakazakii 细胞，需要多次 EMA 处理以避免假阳性结果。我们的快速检测方法也可能提高 2012 年《细菌分析手册》中公布的新 FDA 方法中所需的筛选部分的灵敏度。
